Fig. 2

Confirmation of the Alamut-predicted creation of a new splice donor site by the SPINK1 c.194 + 13T > G variant. a Schemas for the splicing of intron 3 with respect to the wild-type and variant alleles of c.194 + 13T > G. The splice donor (GT) and splice acceptor (AG) signals potentiating the normal and aberrant splicing of intron 3 are highlighted in bold and underlined. The rightward pointing blue arrow indicates the forward allele-specific primer designed to amplify the predicted aberrant transcript (as shown in b). The 12 bp intronic sequence inappropriately included within the predicted aberrant transcript is indicated by a red box. The amino acid sequences of the wild-type and predicted mutant proteins are also shown. b PCR identification of the aberrant transcripts expressed from the HEK293T cells transfected with the c.194 + 13T > G variant-containing maxigene expression construct. The primers used for amplification were the forward allele-specific primer as illustrated in (a) and a reverse primer located within the 3′ untranslated region of the expression vector. No PCR products were identified in cells transfected with the wild-type maxigene expression vector. Plus and minus symbols refer to cells treated with and without cycloheximide, respectively. c Sequence of the c.194 + 13T > G/+ PCR products as illustrated in (b). The 12 bp intronic sequence included within the aberrant transcript is indicated by a red box